Cell and particle counting and measuring instruments, examples being those sold under the trademark Coulter Counter.RTM. by Coulter Electronics, Inc., Hialeah, Fla., employ electronic sensing means which directly respond to the electrical resistance of each cell to count and measure each cell and progressively record cell parameters of a sample of cells suspended in an isotonic electrolyte solution. The Coulter Counter.RTM. particle measuring instruments operate upon the well-known and documented principle of particle and cell measurement employing a sensing aperture path, which also is disclosed in Coulter U.S. Pat. No. 2,656,508 and improvement U.S. Pat. No. 3,259,842.
In the commercial Coulter Counter.RTM. particle analyzer constructed in accordance with the heretofore mentioned U.S. Pat. No. 2,656,508, field excitation has been supplied by a direct current or low frequency source. As previously described, the electrical change caused by the passage of a particle through the electric field of small dimensions, excited by a direct or low frequency current, is approximately proportional to particle size. A direct current is considered to be of zero frequency in this application. However, the impedance sensing principle has been materially expanded to provide information concerning particles being studied, not limited only to characteristics due to the size of particles, but including characteristics due to the composition and nature of the material constituting the particles, as disclosed in U.S. Pat. No. 3,502,974 to Coulter et al. and U.S. Pat. No. 3,502,973 to Coulter et al. These prior art apparatuses generally have at least two current sources, both of which are applied to the sensing zone simultaneously, one having a radio frequency and the other being a "zero frequency" direct current or, alternatively, having a sufficiently low frequency that the reactive part of the particle impedance has a negligible effect on the response of the apparatus. At radio frequencies, the membrane capacitance shunts the cell's membrane resistance so that the high frequency measurement gives a size measurement which is a function of the cell's size and its internal conductance. One of the useful particle descriptors that can be obtained from this dual source arrangement is known in the art as the "opacity" of the particles. With a biological cell, opacity approximately measures the internal conductivity of the cell. Opacity also can be described as measuring the ratio of size as measured at radio frequency to size as measured at low or zero frequency.
U.S. Pat. No. 3,831,087 to Schulz et al., owned by the Assignee of the present invention, shows that as the electrical field strength increases in an electronic particle sensing aperture of the above-described type, electrical breakdown of the cells' membranes will occur at critical field strengths, resulting in the cells having a lower electrical resistance. The breakdown characteristics of cells, processed on a bulk basis, are shown by a graph of apparent mean cell volume (MCV) versus electrical aperture current. The data from processing a blood sample normally defines a breakdown curve having a first leg, the slope of which is representative of the average size and shape of the cells, and a second leg, the slope of which is representative of the average size, shape and internal conductivity of the cells, as shown and described in the Schulz patent. By ratioing the slope of the second leg with that of the first leg, the resulting ratio basically measures the average internal conductivity of the cells. The point of intersection of the two legs is described as the average breakdown point of the cells. The drawback of this technique is that only average values for a batch of cells can be obtained and, with the structure and techniques of the Schulz patent, the cells cannot be individually examined on a cell by cell basis. The average effect eliminates meaningful data on cell characteristics.
U.S. Pat. No. 3,560,847 to Boyd discloses a particle analyzer arrangement wherein the particle suspension through the sensing aperture is stopped momentarily, so that a single particle is in the sensing aperture. With the particle in the aperture, a ramp voltage is applied, allowing the breakdown curve to be generated for a single cell. Although this technique appears to provide breakdown information on a cell by cell basis, it is not practical to use this technique, due to heating of the liquid by the electrical current when stopped in the sensing aperture and further due to the extra difficulty in stopping the fluid stream in the sensing aperture to allow a single particle to be positioned therein. More specifically, although the heating problems are lessened by the short sensing aperture of FIG. 2 of Boyd, the particle alignment problems of having the particle coincident with the sensing aperture are increased. On the other hand, in FIG. 3 of Boyd, the elongated sensing aperture decreases the particle alignment problem, but increases the heating problem.
In an article entitled "Application of the Electrical Sizing Principle of Coulter to a New Multiparameter System", IEEE Transactions of Biomedical Engineering, Volume BME-27, No. 7, July 1980, pp. 364-369, by the Applicant herein, the volume, breakdown voltage and internal conductivity is determined for each cell, thereby overcoming the previously described problems associated with bulk processing and averaging effects therefrom. In this particle analyzer arrangement, a voltage ramp is applied across the orifice as a particle approaches. As the particle passes through the orifice, the field strength increases and breakdown is observed by a change in slope of the ramp signal, the degree of this change yielding the underestimation of the cell volume after breakdown. In order to accommodate the dynamic range of the particle pulse signals, a differential aperture-amplifying system is used and includes a pair of sensing apertures that are fluidly and electrically in parallel, one being used as a reference (no particles pass therethrough) and the other being used for measuring the particles. This same structure and technique is described in U.S. Pat. No. 4,220,916 to Zimmerman et al. This arrangement has the drawback of requiring relatively long orifices or low flow rates to allow enough time for applying the voltage ramp while the particle remains within the sensing aperture. Also, avoiding particle coincidence problems in long orifices mandates very dilute particle suspensions. Additionally, the dynamic impedance of the apertures as a function of applied current or field, as described in the above-mentioned IEEE article, complicates both the measurement and the electronics.
In the above-mentioned article, the phenomenon of the dielectric breakdown of a cell membrane and the consequences of the same are described. Briefly described, dielectric breakdown of a cell membrane is a phenomenon characterized by a reversible increase in the membrane conductance in response to an externally applied electric field pulse across the cell membrane. Once a critical membrane potential, i.e., breakdown voltage, has been reached, the membrane breaks through and becomes permeable to ions. The breakdown probably reflects the elastic and dielectric properties of the membrane. The critical breakdown field is altered in the presence of membrane agents and possibly by diseases. Breakdown in an electronic particle analyzer is detected by the underestimation of size after breakdown has occurred. Due to the increase in membrane conductance, the current lines pass partly through the cell interior, which is more conductive than the original membrane, leading to a reduction in the particle induced signal. The degree of underestimation depends on the internal conductivity and field induced increase in membrane conductance of the cell in relation to the external conductivity.
U.S. Pat. No. 3,793,587 to Thom et al. is of interest for showing back to back particle sensing apertures.
U.S. Pat. Nos. 3,502,973 to Coulter et al. and 3,502,974 to Coulter et al. are incorporated by specific reference herein.